1. Field of the Invention
The present invention relates to a novel kaurene synthase and a DNA which encodes the enzyme.
2. Description of the Related Art
ent-Kaurene is an important intermediate in gibberellin (GA) biosynthesis, and is synthesized from geranylgeranyl pyrophosphate (GGPP) via copalyl pyrophosphate (CPP). These steps are catalyzed by ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively (Coolbaugh, R. C., The Biochemistry and Physiology of Gibberellins, Vol.1, Pracger Publishers, New York, pp.53-98, 1983). Other terpenoids such as carotenoids, xanthophyll, and phytol are also synthesized from geranylgeranyl pyrophosphate as a precursor, while copalyl pyrophosphate is used as precursors for macrocyclic diterpene biosyntheses. ent-kaurene synthase A and ent-kaurene synthase B are thus important enzymes in the initial stage of gibberellin biosynthesis (Coolbaugh, supra; Chung C. H. et al., Plant Physiol., 80, pp.544-548, 1986; Graebe, J. E., Annu. Rev. Plant. Physiol., 38, pp.419-465, 1987).
Duncan et al. separated ent-kaurene synthase A and ent-kaurene synthase B from native cucumber (Marah macrocarpus L.) using a QAE column. The authors suggested that the conversion of geranylgeranyl pyrophosphate to ent-kaurene is catalyzed by the two distinguishable enzymes (Duncan, J. D. and West C. A., Plant Physiol., 68, pp.1128-1134, 1981). In this article, it is shown that ent-kaurene synthase A and ent-kaurene synthase B closely associated with each other during the ent-kaurene synthesis and that ent-kaurene synthase B preferentially utilized endogenous copalyl pyrophosphate produced by ent-kaurene synthase A rather than exogenous copalyl pyrophosphate.
As shown in the scheme set out below, ent-kaurene synthase A catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP) to copalyl pyrophosphate (CPP), and the resulting copalyl pyrophosphate is successively converted to ent-kaurene by ent-kaurene synthase B. ##STR1##
In recent years, cDNA clonings of gibberellin biosynthases have been reported. GAl locus of Arabidopsis was isolated by genomic subtraction (Sun, T. P., et al., Plant Cell, 4, pp.119-128, 1992) and the corresponding cDNA clone was verified to encode ent-kaurene synthase A by over-expression system in E. coli (Sun T. P. and Kamiya, Y., Plant Cell, 6, pp.1509-1518, 1994). Anl locus of maize was cloned by the transposon tagging technique and the deduced amino acid sequence from the cDNA was revealed to have significant homology with Arabidopsis ent-kaurene synthase A, although the function of the expressed protein remains unknown (Bensen, R. J., et al., Plant Cell, 7, pp.75-84, 1995). A putative 3b-hydroxylase in gibberellin biosynthesis was also cloned by the T-DNA tagging method using Arabidopsis GA4 mutant (Chiang, H.-h., et al., Plant Cell, 7, pp.195-201, 1995).
As for ent-kaurene synthase B, the enzyme was partially purified from Ricinus communis (Spickett, C. M., et al., Phytochem., 37, pp.971-973, 1994). Ricinus communis biosynthesizes kaurene and its family diterpenes, i.e., bayarene, torakiroban, and sandarakopymaradiene. These compounds are synthesized through selective cyclizations from a common intermediate, copalyl pyrophosphate. Although a cloning of an ent-kaurene synthase B gene was attempted (15th International Conference on Plant Growth Substance, Jul. 14-18, 1995, Minneapolis, USA, Kamiya, Y., et al., Subject No. 040 and Saito, T., et al., Subject No. 110), the complete sequence of a cDNA and the amino acid sequence of the enzyme has not been reported.